Enkephalin and Beta-endorphin opioid peptide neurotransmitters are synthesized as proenkephalin (PE) and proopiomelanocortin (POMC) precursors, respectively, which require proteolytic processing in secretory vesicles to form active neuropeptides. The extent of PE and POMC processing in chromaffin cells and pituitary, respectively, is limited and varied. These observations suggest that endogenous protease inhibitors may be involved in proneuropeptide processing. Therefore, this study has investigated the hypothesis that endogenous protease inhibitors maybe colocalized with, and inhibit, pro-neuropeptide processing enzymes. Our molecular studies have established two novel, secretory vesicle protease inhibitors, endopin 1 and 2 that posses distinct target protease specificity. These two endopins share homology with the serpin family of protease inhibitors, and possess active sites consistent with pro-neuropeptide processing. Endopins are colocalized with, and inhibit the PE processing enzyme PTP ('prohormone thiol protease'); endopins also inhibit POMC cleaving activity in pituitary secretory vesicles. In continuing studies, the goal of this proposal will be to compare the roles of endopin 1 and 2 in the proteolytic processing of the pro-neuropeptides PE and POMC. In specific aim 1, active-site mutants and chimeras of endopins will define structural features of the reactive site loop responsible for inhibition. Inactive mutants will be used as controls in endopin expression studies (aims 2, 3, 5). In specific aims 2 and 3, regulation of PE and POMC processing by endopins will be assessed in primary cultures of chromaffin and pituitary cells, respectively, by endopin sense and antisense expression. The tissue distribution of the endopins and their subcellular localization in neuropeptide containing secretory vesicles will be studied in specific aim 4 by confocal immunofluorescence and immunoelectronmicroscopy. Endopins 1 and 2 inhibit PE and POMC processing activities in secretory vesicles, suggesting target proteases of endopins in these vesicles. Because secretory vesicle endopins form complexes with proteases, endopins can be used as a tool for molecular cloning of endopin-interacting protease(s) from pituitary and chromaffin cells (aim5). Evaluation of an endopin-interacting clone for enhanced PE and POMC processing, regulated secretion, and inhibition by endopin(s) will lead to consideration of the clone as an endopin target protease. This study will define a new, endogenous serpin protease inhibitor mechanism for regulating peptide neurotransmitter production. Results will indicate the role of endopins in processing PE and POMC into active enkephalin and Beta-endorphin opioid peptides, studied here as model peptide neurotransmitters. Serpin regulation of opioid peptides that mediate analgesia and stress may lead to development of new therapeutic approaches in pain and neurologic disease.